4,4'-Methylene-bis(2-chloroaniline) (MCA) is an industrially used aromatic amine which is carcinogenic for rats, mice, and dogs. It is not known whether MCA is carcinogenic in humans, but it bears a structural resemblance to other aromatic amines which are known to be potent human urinary bladder carcinogens. Little is known regarding the metabolism of MCA or the mechanisms by which it induces tumors. The objectives of this project are to examine the metabolic activation pathways for MCA in rats and to make a species comparison of the urinary metabolites of MCA in rats and humans. To facilitate the isolation and identification of metabolites, methods for chemical synthesis of anticipated MCA metabolites will be developed; isotopically labeled derivatives will be used for metabolism and binding studies in rats. In vitro studies with rat liver subcellular fractions will be conducted to determine whether known pathways for metabolism of aromatic amines apply to MCA (i.e. N-acetylation, N-hydroxylation, and activation for macromolecular binding by N,O-acyltransferase or sulfotransferase). The possibility that oxidation of the methylene bridge leads to reactive benzylic derivatives will also be explored because our preliminary experiments have suggested that cleavage at the methylene bridge is a significant pathway in rats in vivo. Urine from MCA-treated rats and from workers exposed occupationally to MCA will be analyzed for the presence of metabolites which represent activation and/or detoxification pathways; analytical techniques will include high pressure liquid chromatography and derivatization followed by gas chromatography/mass spectrometry. These experiments should provide basic information regarding the mechanisms by which MCA may induce tumors in animals; they should also indicate the occurrence of activation/detoxification pathways in humans and aid in the evaluation of exposure to MCA.